Process for the production of collagen fiber fabrics in the form of felt-like membranes or sponge-like layers

ABSTRACT

PRODUCTION OF FELT-LIKE MEMBRANE OR SPONGE-LIKE LAYERS OF COLLAGEN FIBERS BY DECOMPSING SKIN/OR TENDONS OR OTHER ANIMAL CONNECTIVE TISSUES RICH IN COLLAGEN UNDER ALKALINE AND/OR ACID CONDITIONS; MECHANICALLY COMMINUTING THE DECOMPOSITION PRODUCT; SUSPENDING THE COMMINUTED COLLAGEN STOCK OBTAIN IN WATER TO FORM A HOMOGENEOUS COLLAGEN SLURRY; ADDING A TANNING OR CROSS-LINKING AGENT TO THE SLURRY; FOAMING THE COLLAGEN SLURRY; FREEZING THE FOAMED COLLAGEN SLURRY SO OBTAINED IN THE FORM OF A LAYER AT -5 TO -40* C.; INCUBATING THE FROZEN SLURRY FOR 1 TO 30 DAYS; AND THEN FREEING THE BULK OF THE WATER THEREFROM BY SIMPLE MECHANICAL SQUEEZING AND.OR EVAPORATIVE DRYING.

1974 M. CHVAPIL 3,823,212

PROCESS FOR THE PRODUCTION OF COLLAGEN FIBER FABRICS IN THE FORM OFFELTLIKE MEMBRANES OR SPONGE-{11KB LAYERS 2 Sheets-Sheet L N H U I g u mZOC. mDUZ WEDOI mm H July 9 Filed May 28. 1971 NBEDVW'IOI) '9/9'NOLLdk-JOSQV HELLVM FIG. 2.

y 9, 1974 M. CHVAPIL 3,8233%2 PROCESS FOR THE PRODUCTION OF COLLAGENFIBER FABRICS I THE FORM OF FELT'LIKE MEMBRANES 0R SPONGE-LIKE LAYERSFiled May 28. 1971 2 Sheets-Sheet 2v l O N 9 (NBDVT'IOI) .LHOIBM AUG 9/9BILLY/M) AllDVdVl) NOLLdHOSBV HELLVM United States Patent O 3,823,212PROCESS FOR THE PRODUCTION OF COLLAGEN FIBER FABRICS IN THE FORM OFFELT-LIKE MEMBRANES OR SPONGE-LIKE LAYERS Milos Chvapil, Tucson, Ariz.,assignor to Flrma Carl Freudenberg, Patent Abteilung, Weinherm, GermanyContinuation-impart of abandoned application Ser. No. 878,118, Nov. 19,1969. This application May 28, 1971, Ser. No. 148,116 Claims priority,application Germany, Nov. 27, 1968, P 18 11 290.8-44 Int. Cl. 329d 27/03US. Cl. 264-49 17 Claims ABSTRACT OF THE DISCLOSURE Production offelt-like membranes or sponge-like layers of collagen fibers bydecomposing skin and/or tendons or other animal connective tissues richin collagen under alkaline and/or acid conditions; mechanicallycomminuting the decomposition product; suspending the comminutedcollagen stock obtained in water to form a homogeneous collagen slurry;adding a tanning or cross-linking agent to the slurry; foaming thecollagen slurry; freezing the foamed collagen slurry so obtained in theform of a layer at 5 to -40 C.; incubating the frozen slurry for l to 30days; and then freeing the bulk of the water therefrom by simplemechanical squeezing and/ or evaporative drymg.

This application is a continuation-in-part of Application Ser. No.878,118, filed Nov. 19, 1969, now abandoned.

This invention relates to the production of collagen fiber structures.It more particularly refers to an improved process of producing suchstructures which are felt or foam-like and which are more economicalthan prior art processes.

It is known to use collagen fiber fabrics, referred to as collagensponges, in medicine, in particular in surgery, as plugs and/ orbandaging material for staunching bleeding. The production of suchcollagen sponges is described, for example, in German Patent ApplicationS 97,055 and in French Pat. 1,441,817. As a rule, this known process forthe production of collagen fiber fabrics comprise decomposing cattleskin and/ or cattle tendons under alkaline and/or acid conditions;mechanically comminuting the decomposition product whereby the collagenstock is obtained; suspending such in water to form a homogeneouscollagen slurry; freezing the collagen slurry obtained in the form of alayer after adjustment of the pH value to about 7.0; and removing thebulk of the water from the frozen collagen layer by lyophilisation or byextraction with organic solvents.

This process has the advantage that there can be incorporated into thecollagen slurry compounds which, in an advantageous manner, affect theproperties of the collagen fiber fabrics produced therefrom. Forexample, it is possible to incorporate plasticizers, as well astherapeutically and/or diagnostically active compounds, for instancecompounds inhibiting or promoting clotting of the blood, analgetics,antibiotics or compounds containing isotopes, and in this way producecollagen fiber fabrics in the form of sponge-like fabrics which can beused for many purposes.

A disadvantage, however, is that this known process is relativelycostly, more than 80% of the costs incurred being as a rule accountedfor by the stage of the process in which the bulk of the water presentis removed from the frozen collagen layer, i.e. the cost of energy forthe lophilisation or the cost of solvent for the extraction makes icethe process inordinately costly and has a very detrimental effect on theeconomy thereof.

A further disadvantage is that while the known process makes possiblethe production of collagen fiber fabrics in the form of sponge-likelayers, it does not, however, permit the production thereof in the formof felt-like membranes of small thickness and adequate water absorptioncapacity. There is a great need for such membranes, however.

Membranes or films of this kind can find varied use, for example, inmedicine, for instance for the treatment of skin burns and abrasions,where large wound areas must sometimes be covered, in order to protectthe injured areas from additional external injury, prevent infection ofthe wound from occurring and prevent bandaging material from sticking tothe wound. Further, such felts can promote healing of the wound bysupplying therapeutically active compounds, and can also preventmaceration of the wound by slow absorption of the exudation formed.

Still further, such felt membranes may be employed as substitutes orreplacements for certain body membranes, for example the dura mater.Heretofore, only the membranes obtained from newborn infants andconstituting the allantois, the amnion and the chorion, as well as thedura mater recovered from corpses, have been available for this purpose,or alternatively physically foreign films or foils, for example tantalumfoils, which are not absorbable and must be removed in a secondoperation, have had to be employed. Moreover, the membranes heretoforeemployed for the purposes indicated have the disadvantage that theycannot practically be charged with therapeutically active compounds forpromoting the healing of wounds. Also, as a rule, they are not capableof absorbing the exudation that is formed by the wound. Additionaldisadvantages are that the production of such prior art membranes istime-consuming and costly and that it is difficult to produce suchmembranes which are free from antigenic effects and have reproducibleproperties.

:One object of this invention is, therefore, to provide a process, whichis simple and economically attractive, by which collagen fiber fabricscan be produced in the form of felt-like membranes or sponge-like layershaving high mechanical strength and at the same time excellentflexibility.

Another object is to produce such fabrics in such a manner as to impartthereto a high capacity for absorbing liquids, a limited antigeniceffect, a high binding capacity for solids and a fibrillogenesisstimulating action.

Still another object of this invention is to provide a novel process forproducing collagen felts or sponges which is economically attractive.

Other and additional objects of this invention will be come apparentfrom a consideration of this entire specification including the claimsand drawings hereof.

In accord with and fulfilling these objects, one aspect of thisinvention lies in the surprising discovery that a foamed collegan slurrycontaining appropriate known tanning or cross-linking agents becomescross-linked below a certain minimum temperature below the freezingpoint of the slurry with time to a felt-like or sponge-like structure.If, therefore, a foamed collagen slurry containing tanning agent isfrozen in the form of a layer below a particular maximum temperature andis then incubated at such low temperatures for a given amount of time, asponge or felt form product results. The structure of the product can becontrolled by adjusting the incubation time and incubation temperaturerelative to one another so that felt-like membranes or sponge-likelayers of only moderately swellable collagen fibers which are onlypartially cross-linked, or of greatly'swellable collagen fibers whichare three-dimensionally cross-linked to a high degree respectively, areformed. The frozen mass is mostly water, e.g. up to about 99 volumepercent. The bulk of this water frozen with the collagen fiber fabric isnot removed from the structure in the frozen state as in the prior art,but rather the structure is thawed and the water removed by mechanicalsqueezing and/or conventional evaporation drying techniques at room temperature. This results in a marked economic advantage since it is notnecessary to use lyopilization or freezedrying or solvent exchangetreatments as in the prior art.

The industrial utility of the simply and cheaply produced collagen fiberstructures according to this invention extends not only to the medicalfields which utility is generally known (see above), but also tonumerous commercial fields where an absorbent and wear-resistantmembrane or layer of good strength and flexibility, which can becombined if necessatry with plastics or textile materials, is desired,for instance as leather substitute material, for example in themanufacture of lining leather, or in other commercial fields, forexample for making sanitary napkins and/or tampons.

The invention therefore encompasses an improved proc ess for theproduction of collagen fiber structures, in the form of felt-likemembranes or sponge-like layer with controlled structures and controlleddegrees of swellability. In the process animal skin and/ or tendons and/or other animal connective tissues rich in collagen is decomposed underalkaline and/or acid conditions; mechanically comminuted; the comminutedcollagen stock obtained suspended in water to form a homogeneouscollagen slurry with tanning agent added to such slurry and if necessaryand/or desirable with the simultaneous addition of plasticizers and/ortherapeutically or diagnostically active compounds; and the collagenslurry thus obtained is frozen, incubated, thawed and then freed fromthe main portion of water retained therein, which process improvement ischaracterized in that the comminuted collagen mass is made into ahomogeneous collagen suspension in water and foamed in the presence ofair and/or an inert gas, with the collagen suspension having a collagencontent of about 0.3% to 3%, preferably from about 0.5 to 2% (dry weightbasis), and an adjusted pH value of 3 to 5.5, preferably to 4 to 5; thefoamed collagen suspension is frozen in the form of a layer attemperatures from --5 to 40 C., preferably between to 30 C., and allowedto stand for about 1 to 30 days, preferably from about 2 to 8 days,under atmospheric pressure at the indicated temperatures, thereafter thefrozen collagen layer is thawed at temperatures of about 10 to 30 C.,preferably at room temperature, and the main portion of the water inthis structure is removed therefrom by mechanical squeezing. If desired,the squeezed-out collagen fiber structure can be further dried at roomtemperature.

In the accompanying drawing:

FIG. 1 is a graphical representation showing the effect of incubationtime on the welling (0.9% solution of sodium chloride in waterabsorption) capacity of a collagen fiber structure made according tothis invention; and

FIG. 2 is a graphical representation showing the swelling (waterabsorption capacity) of collagen fiber structures made according to thisinvention compared with those made according to the prior art.

In accordance with a preferred embodiment of the process of theinvention, first a collagen mass obtained through alkaline and/or aciddecomposition of beefhide or beef-tendons is mechanically comminuted andthe thus obtained collagen mass washed several times with a 10% sodiumchloride solution in order to remove impurities which as a rule areresponsible for the antigeneous effect of impure collagen. The washedcollagen mass is then suspended in water and the pH value adjusted toabove 4, preferably from 4 to 5. This exit collagen mass, with acollagen content of about 8 to 13% (dry weight basis), is then treatedwith so much water and with tanning materials, as well as plasticizersand/or therapeutically or diagnostically effective compounds, asnecessary or desired, that the collagen content of the obtained mixture(dry weight basis), is 0.3 to 3%, preferably 0.5 to 2% whereupon theobtained mixture is foamed as aforesaid, preferably in a mixingapparatus running at high speed at about 500 to 3,000 r.p.m. and finallythe foamed collagen slurry obtained is poured into containers of, forexample, plastic, glass or metal in the form of layers about 0.5 to 5cm. thick, preferably 0.5 to 3 cm. thick, and frozen in the mannerindicated and incubated at said temperatures, after which incubation thefrozen layers are thawed out, preferably at room temperature, and watermechanically squeezed thereout of. The squeezedout collagen fiberstructures are further dried by evaporation if necessary at room:temperature or above and/or, possibly by blowing a stream of air on toone or both sides of the material to be dried. For preventing distortion, it is expedient to cover the collagen fiber structure to be driedon both sides with an absorbent web, for example filter paper, andarrange thereover a strong screen to preserve the dimensional stabilityof the material to be dried. The dried collagen fiber structure, whichas a rule still contains about 10% of water, can then be cut into piecesof suitable size and shape, sealed in plastics films and sterilized.

As already mentioned, in order to carry the process of the inventioninto effect, it is important that the collagen slurry intended to befrozen is foamed, so as to obtain a slurry permeated by numerous gasbubbles. It has proven to be advantageous to homogenize the collagenslurry in a mixing apparatus operated at about 500 to 3000 r.p.m. whilesimultaneously introducing into the slurry air or inert gases, forexample nitrogen or carbon dioxide. It has been found that a collagenslurry foam with a viscosity of about 200 to 600 cp., preferably about300 to 500 cp., can be worked up in a particularly advantageous mannerinto high-grade collagen fiber structures. The viscosity can becontrolled in an advantageous manner by adjusting the pH value or thecollagen content, a col.- lagen slurry with a comparatively low pH valueexhibiting as a rule a comparative high viscosity.

The process of the invention can be controlled in such manner as toproduce collagen fiber structures which are cross-linked to a greater orlesser degree and have a greater or lesser capacity forabsorbing'liquids or fluids. The fluid absorption capacity of thecollagen fiber structures obtained depends, assuming a given proportionof tanning agent, primarily on the incubation time and incubationtemperature.

It has proven to be advantageous to adjust the three indicated factorsaffecting the structure of the collagen fiber structures obtained,namely incubation time, incubation temperature and tanning agentcontent, relative to one another on the basis of preliminary tests. As arule, a collagen slurry containing about 1% of glutaraldehyde, referredto total volume of the collagen mixture, as tanning agent produces, withincubation for about 8 days at 8 C., a collagen sponge having athree-dimensional net structure which is developed to such an extentthat, after absorbing water, it assumes its original form again on beingwrung out. In contrast, the same material otherwise treated in the sameway but after incubation for about 8 days at 2 C. yields a collagenproduct in the form of a gel having a comparatively low degree ofcrosslinking and water absorbability.

In order to carry out the process of the invention, conventional tanningagents, known as hardening agents for proteins, may be incorporated intothe collagen slurry. The use of glutaraldehyde or melamine formaldehyderesins, which are physiologically harmless, has proven advantageous. Thetanning agents may be employed in various concentrations. Concentrationsof about 0.1 to 3%, preferably 0.2 to 2% referred to the total volume ofthe collagen slurry, are suitable.

The addition of plasticizers to the collagen slurry, in particular whenit is intended to produce collagen fiber structures in the form offelt-like membranes, is ad- 'vantageous for controlling the elasticproperties of the collagen fiber structures produced by the process ofthe invention. The known conventional plasticizers, for instanceglycerine or sorbitol, may be incorporated into the collagen slurry. Theplasticizers may be employed in various concentrations. Concentrationsof about 0.1 to 3%, preferably 0.3 to 1%, referred to the total volumeof the collagen slurry, are suitable. If the plasticizers are present inconcentrations higher than those indicated, undesirably stronglyhydrated collagen fibers of only low tensile strength may result.

If therapeutically active compounds are to be incorporated into thecollagen slurry, these may be known conventional compounds usuallyemployed in wound dressings and surgery, for example compounds useful ininhibiting or promoting blood clotting, disinfectants, analgesics,antibiotics, for instance bacitracin, neomycin or tetracyclin, and thelike. Diagnostically active compounds may be added to the collagenslurry, such as compounds containing radioactive isotopes. Thetherapeutically and/ or diagnostically active compounds may be employedin various concentrations. It has proven to be advantageous to determinethe appropriate concentration by preliminary tests in the known matter,so as to prevent the collagen proteins being precipitated where suchcompounds are used in too high concentrations. When antibiotics areemployed, concentrations of about 20 to 100%, preferably 30 to 60%,referred to the dry weight of the collagen present in the collagenslurry, are advantageous.

In order to carry out the process of the invention, it has been found tobe advantageous to remove the bulk of the water from the final productmechanically by squeezing such out from the collagen layers thawed outat temperatures of about to 30 C., preferably at room temperature. Thissqueezing may be effected by known conventional methods, for example byplacing the collagen fiber fabric on a firm support and then pressingsuch with a rubber squeegee, or alternatively by pressing such betweentwo rotatably arranged pressure rolls, having an adjustable nip.Depending on the thickness of the collagen fiber structure, a nipspacing of about 0.2 to 0.5 cm., preferably 0.3., between the twopressure rolls is suitable.

If the finished collagen fiber structures are to be sterilized, ifnecessary after cutting into pieces of suitable shape and size and aftersealing in a plastic film, for example polyvinyl chloride film,sterilization may be by means of Co isotopes, for example in a dosage of2 to 4 mrad., or by means of ethylene oxide gas, which have proved to beadvantageous, since in this way the properties of the collagen fiberstructures are not detrimentally alfected.

According to one particularly advantageous method of carrying out theprocess of the invention, the collagen slurry, containing the indicatedadditions if necessary, is poured into containers into which a textilefabric of naturally occurring or synthetically produced staple fibersand/or substantially continuous mono or multifilaments has beenpreviously introduced. This is then processed further as has beenindicated above. The products produced in this way are combinations ofcollagen fibers and the other fibers utilized and are particularlyadvantageous for treating wounds to which bandaging materials stickeasily.

The following non-limiting illustrative Examples are intended toillustrate the invention.

Example 1 500 ml. of cold water were added to 100 g. of a collagenslurry, having a collagen content of 8% (dry weight basis). The mixturethus obtained was homogenized in a mixing apparatus at 800 r.p.m. toform a collagen slurry foam while slowly adding 20 ml. of glycerine and2 grams of glutaraldehyde. The foamed collagen slurry obtained waspoured into trays in the form of layers 1 cm. thick, frozen at 20 C. andleft for 3 days at the temperature indicated. The frozen collagen foamlayers were thereafter allowed to thaw out at room temperature and thenimmediately freed from water by squeezing between two pressure rollswith a nip spacing of about 0.3 cm. The squeezed-out collagen fiberstructure which resulted was dried at room temperature between twofilter papers each having a metal screen placed thereon. The physicalproperties of the felt-like membrane obtained were tested.

The results obtained are given in the following Table:

TABLE 1 Thickness of the felt-like collagen membrane 0.3 cm. Collagencontent, (dry weight basis) 84.5%.

Swelling in 0.9% Na Cl after 24 hours 4.82 times the weight (original).Swelling in 0.07 N HCl after 24 hours 8.60 times Tensile strength of astrip the original weight. 0.2 x 1 x 4.5 cm. 4.2 kg.

Shrinking temperature C.) (for a shrinking temperature of 48 for theinitial collagen 68- 2.

The felt-like membrane produced was sealed in a polyvinyl chloride filmpouch and sterilized with Co isotopes at a dosage of 2.5 mrad.

Example 2 The procedure described in Example 1 was repeated in threetest mixtures, with the difference being that 0.5 g. of the antibioticsbacitracin, neomycin or tetracyclin, respectively, was added to thecollagen slurry during the homogenization. correspondingly advantageousresults were obtained.

Example 3 The procedure described in Example 1 was repeated in two testmixtures, except that a stream of air or nitrogen was introduced intothe collagen slurry during the homogenization. correspondinglyadvantageous results were obtained.

Example 4 The procedure described in Example 1 was repeated, except thatthe foamed collagen slurry was poured into containers on the bottom ofwhich a textile fabric consisting of bandaging gauze had been placed.Correspondingly advantageous results were obtained.

Example 5 Example 6 This Example shows the effect of the concentrationof an added tanning substance on the collagen content and the swellingcapacity of the resulting collagen fiber structure ready for use. Ineach case to 30 gms. of initial collagen slurry, having a collagencontent of 8.5% (dry weight basis), there was added the amount ofglutaraldehyde stated in Table 2 below and the mixture was worked upfurther as previously indicated. The foamed collagen slurry wasincubated for 6 days at -20 C. The results obtained are compiled in thefollowing Table 2.

TABLE 2 Swelling after 24 hours in- Collagen content percent 0.9% NaCl0.01 N H01 Glutaraldehyde added (dry weight as 25% solution (ml.) basis)Times the origlnal weight Example 7 This Example shows the effect of theconcentration of plasticizer on the water content to be calculated onthe basis of the collagen content and on the swelling capacity of theproduct collagen fiber gauze ready for use.

1 ml. of 25% glutaraldehyde and the respective amounts of glycerinestated in Table 3 below were added to 30 g. in each case of the initialcollagen slurry indicated by the method described in Example 1, theslurry having a collagen content of 8.5% (dry weight basis), after whichprocessing was continued in the manner indicated above. The foamedcollagen slurry was incubated for 6 days at 20 C. The results obtainedare compiled in Table 3 hereunder.

TABLE 3 Collagen content, Swelling after 24 hours inreferred to dry 0.9%NaCl 0.01 N H01 substance Glycerine added (ml.) (percent) Times theoriginal weight;

Example 8 This Example shows the effect of the incubation time at 20 C.on the swelling capacity of the produced collagen fiber structure, readyfor use, in 0.9% NaCl solution.

100 g. in each case of the initial collagen slurry indicated were workedup in the manner stated by the method described in Example 1, exceptthat the foamed collagen slurry was incubated for different lengths oftime at 20 C. Thhe swelling capacities of the collagen fiber structure,obtained was tested. The results obtained were plotted in the graph ofFIG. 1. The incubation at 20 C. was 55 hours for the sample marked A, 71hours for the sample marked B and 121 hours for the sample marked C.

The results show that the swelling capacity of the finished collagenfiber structures increases with increasing incubation time.

Examples 9-20 In each example the collagen slurry was prepared in anidentical manner as follows:

100 grams of aqueous acid swollen collagen containing dry collagensolids was mixed with 500 ml. of water with continuous stirring. Thisslurry was placed in 5 liter capacity Waring Blender and another 500 ml.of ice cold water, containing 4 ml. of a 25% aqueous solution ofglutaraldehyde, was added under stirring at 3000 r.p.m. A foamedcollagen slurry having a pH of 3 to 3.5 resulted to which was addedsufiicient 2 normal NaOH to raise the pH to 4.5 to 5 (about 1.5 to 2 ml.of caustic added) to a gel-like consistency. The thus formed gel wastransferred into 12 Petri dishes each of which was 2 cm. high and 15 cm.in diameter.

Six of the thus filled Petri dishes, marked A, were stored for varyinglengths of time at 0 to 2 C. and the other six Petri dishes marked B,were stored for varying lengths of time at C. At the end of the timesspecified below, each Petri dish was removed from the freezer,

thawed at room temperature and then squeezed for 5 minutes under a 50kg. load. Each product was dried at room temperature for a constantweight, cut into 50 mg. pieces and then immersed in water at 20 C. for 5minutes. The average amount of water absorbed and the mean deviation wasdetermined and is reported per gram of sample (dry weight).

Additionally, each sample was tested to determine the time in seconds itlook for it to be completely wetted with such 20 C. water.

The results are set forth in the following table:

TABLE 4 Incubation Water time absorp- Wetting Sample (days) Productappearance tion time A (2 C.) 1 Highly swollen g l 0 0 A (2 C.) 3 ..do 00 A (2 C.) 5 do 0 0 A (2 C.) 7 d0 0 0 A (2 O.)- 10 do. 0 0 A (2 C.) 15do 0 0 B (20 C.) 1 Sheet 2 mm. thick. 5:0. 8 120 B (--20 C.) 2 Felt lIDJIl. thick 8 1. 2 68*24; B (20 C.)... 3 Sponge 4 mm. thic 16*4 25*7 B(20 C.) 4 Sponge 12 mm. thick--- 38= =9 12 =2.4 B (20 C.) 5 Sponge 18mm. thick 47= =8 7:1.4 B (--20 C.) 6 do 486 5. 2 =1.2

Example 21 500 ml. of water, 2 ml. of sorbitol and 3 ml. of 25glutaraldehyde were added to g. of the initial collagen slurryindicated, having a collagen content of 13.5% (dry weight basis), andthe mixture was suspended in a mixing apparatus at 800 r.p.m., with theformation of foam, to form a homogeneous collagen slurry foam. The pHvalue of the formed collagen slurry was adjusted to 5 by the addition ofsaturated sodium bicarbonate solution. The collagen slurry foam obtainedwas frozen at 8 C. in the form of a layer and was incubated for 8 daysat this temperature. The collagen layer was thereupon allowed to thawout at room temperature, after which the water was mechanically removedby squeezing it out of the thawed-out collagen fiber structure. Thesqueezed-out collagen fiber structure was dried between two sheets offilter paper and two screens at room temperature. About 1 sq. m. ofcollagen fiber structure in the form of a sponge-like layer 0.5 cm.thick and having a water content of about 10% was obtained.

Example 22 4.5 liters of water, 7.5 ml. of sorbitol, 10 ml. of 25glutaraldehyde and 10 ml. of 1.5 N hydrochloric acid were added to 500g. of the initial collagen slurry indicated, having a collagen contentof 13% (dry weight basis), and the pH value of which was 4.5, afterwhich the mixture obtained was mixed for 3 hours in a mixing apparatusat slow speed. 30 ml. of saturated sodium bicarbonate solution wereadded to the mixture obtained in order to lower the viscosity of themixture. The mixture obtained was then homogenized in a mixing apparatusat a high speed of 600 r.p.m. with the formation of foam. The foamedcollagen slurry was incubated for 14 days at 8" C. It was further workedup the method described in Example 9. Correspondingly advantageousresults were obtained.

Example 23 The method described in Example -9 was repeated, except that3 g. of bacitracin, in the form of a powder, were added to the collagenslurry during the homogenization. Correspondingly advantageous resultswere obtained. The blood-clotting action of the tanned collagen fiberfabric may be attributable to the fact that, on contact of the bloodwith the large sponge-like surface of the collagen fiber structure, thethrombocytes of the blood burst by reason of the local change inviscosity. It may also be attributable to the fact that the blooddissolves part of the collagen sponge with the irreversible formation ofa gel,

so that a uniform collagen coating is formed over the bleeding woundwhich exerts a plug affect.

Example 24 This Example shows the effect that the water present in thefrozen collagen layer exerts on the development of the net, cross-linkedstructure of the collagen fiber structure.

- In comparison tests, a collagen slurry of the composition described inExample 9 was frozen at -20 C. in the form of a layer, following whichthe bulk of the water present was removed from the frozen collagen layerby the known prior art technique of lyophilization. In another series oftests, the collagen slurry was frozen at -20 C. by the process of theinvention and incubated for 8 days at the temperature indicated withoutremoval of the water present in the frozen layer. The collagen fiberstructures obtained were tested for their water absorption capacity. Theresults obtained were plotted in the graph shown as FIG. 2.

The results show that the collagen fiber structures produced by theknown prior art process has a lower initial water absorption capacitythan the collagen fiber structures produced by the process of theinvention, and also that after a longer time the water absorptioncapacity of the two samples is substantially equal.

Example 25 250 g. of initial collagen slurry (13% of collagen, dryweight) were mixed with 1.7 liters of water, ml. of sorbitol (availablecommercially under the name Karion F) and 8 ml. of 25% glutaraldehydesolution, the mixture was foamed after which the foamed collagen slurrywas frozen at 8 C., in the form of a layer 1 cm. thick, and incubatedfor 3 days. After thawing out, the water was mechanically squeezed out.The collagen fiber structure obtained was a collagen sponge about 1 cm.thick which had the following properties:

Water content 77%. Collagen content, referred to dry weight 23%.

Water absorption, referred to moist weight of the colla- 400 g. H 0/100g. of gen collagen.

Water absorption, referred to dry weight of the collagen 2,000 g. H0/100 g. of

collagen.

The squeezed-out collagen fiber structure was mechanically squeezed outstill further under a pressure of 250 atms. The membrane-like layerobtained was sterilized and stored under sterile conditions. When thismembranelike layer was wetted with water, it absorbed the waterimmediately and rapidly, reforming the original layer 1 cm. thick.

Certain comparative tests have been conducted for the purpose ofestablishing the criticality of both the freezing temperature and theincubation period on the physical properties of the products produced.

Examples 26-27 These Examples illustrate the fact that collagen spongeprepared by the procedure of this invention does not contain any freetanning agent as compared with collagen sponge made by prior artprocesses such as lyophilization processes.

A collagen slurry was prepared with stirring according to the techniquedescribed above containing 100 grams of collage, 1000 grams of water and4 grams of 25% aqueous gluataraldehyde.

The slurry was foamed as aforesaid and separated into two ('2) aliquotseach of which was frozen at 20 C. One of the samples A was maintainedfrozen for 4 hours at 20 C. and then subjected to freeze drying in avirtis freeze drier by heating to 20 C. under a vacu- TABIJE 5 SampleMg. glutaraldehyde/ 9 g. A 4417. B None detected.

North-Maximum glutaraldehyde possible in sponge is 66 mg.

These last two Examples point up one very real advantage of the collagensponge made according to this invention as compared to collagen spongesmade by a lyo'philization technique. Sponges made according to thisinvention can be water washed free of tanning agent in the dry spongewhere'as sponges made by a lyophilization technique cannot be re-wetonce they are dried.

What is claimed is:

1. In the production of a collagen fiber structure by forming anacidified slurry of collagen in water containing about 0.3 to 3%collagen on a dry basis and containing about 0. 1 to 3% based on the dryweight of collagen of a tanning agent therefor; freezing said slurry;and removing the water from said frozen slurry; the improvement whichcomprises freezing said slurry at a temperature of up to about 5 C.,maintaining said slurry in the frozen condition for at least about 1day, thawing said frozen slurry, and removing the water from said thawedmaterial.

2. The improved process claimed in claim 1 including freezing at about 5to -40 C. for about 1 to 30 days, thawing at about room temperature andmechanically squeezing the water out of said thawed product.

3. The improved process claimed in claim 1 including foaming said slurrybefore freezing such and (freezing such in foam form.

4. The improved process claimed in claim 1 including adjusting the pH ofsaid slurry to about 3 to 5.5 prior to freezing.

5. The improved process claimed in claim 1 including beating asubstantially inert gas into said slurry prior to freezing.

6. The improved process claimed in claim 1 including adjusting theviscosity of said slurry to about 300 to 500 cp. before freezing.

7. The improved process claimed in claim 1 where said tanning agent is amember selected from the group consisting of glutara-ldehyde andmelamine-formaldehyde resin.

8. The improved process claimed in claim 1 including adding about 0:1 to"3% of plasticizer, based on the total volume of collagen slurry, tosaid slurry.

9. The improved process claimed in claim 1 including adding about 10 tobased on the dry weight of collagen, of at least one member selectedfrom the group consisting of a blood clotting inhibitor, a bloodclotting promotor, an analgesic agent, and an antibiotic to said slurry.

10. The improved process claimed in claim 1 including forming saidslurry into a layer about 0.2 to 5 cm. thick prior to freezing such.

11. The improved process claimed in claim 2 wherein said mechanic'alsqueezing is carried out between a pair of rolls having a nip of 0. 2 to0.5 cm.

12. The improved process claimed in claim 1 including admixing saidslurry with a mass of preformed fibers prior to freezing.

13. The improved pr'ocess claimed in claim 1 including freezing at '10to 30 C. for 2 to 8 days and thawing at 10 to 30 C.

14. The improved process claimed in claim 1 wherein said collagenrepresents 0.5 to 2 weight percent of said slurry and said tanning agentrepresents 0.2 to 2 volume percent of said slurry.

15. A substantially tanning agent free collagen fiber structure made bythe process of claim 1.

16. The improved process claimed in claim 1 wherein 10 References CitedUNITED STATES PATENTS 111M964 Artandi. 106-122 10/196 9 Battista 264284/1936 Hill 91--68 5/1935 Sheppard et a1. 87--'Z FOREIGN PATENTS 1/1967Great Britain 26'449 M. J. WELSH, Primary Examiner US. Cl. X.R.

' wig g g u v UNITED S'lATES PATENT oFmu';

' 5 CERTIFICATE OF CORRECTION Patent NOJ 3,823,212 Dated "July 9,"119-74 Inventoflkx M1108 Chvapil I It 'is 'certified that errorappearsin the ohmic-identified patent I and that said Letters Patent arehereby corrected as shown below:

' co1iinm 3, line 17 "mace-$983317" should be "necessary" Colnmn 3 line57 elling" should be f'swelling". Column 6, line 21 "0.07' shou1d be"0.01"

Column 6, line 22 The line "Tensile strength of a strip" should bedropped down to go together with "0.2 x l x 4.5 cm."

Column 7', line 48 change "thhe" to "the" Signed and sealed this 8th dayof October 1974.

(SEAL) Attest:

McCOY M. GIBSON JR. C. MARSHALL DANN Attesting Officer Commissioner ofPatents

